Abstract:

The instant invention relates to systems of reagents and methods for the
detection of chemical and biological targets in a sample. Some
embodiments comprise methods for automatically selecting a set of
reagents to detect one or more targets in a sample, wherein the set of
reagents comprises at least two layers for detection of a first target,
and at least two layers for detection of a second target, wherein the set
comprises reagents that are at least partially redundant. In some
embodiments, the redundancy is created by at least one degenerate reagent
such that the reagent may interact specifically with more than one other
component of a detection system or sample. In some embodiments, the
system or method also includes reagent containers with a
computer-generated code which may further serve to match targets to
appropriate reagents.

Claims:

1. A system for detecting at least two targets in a sample, the system
comprising:(a) a sample potentially comprising at least two targets;(b) a
set of reagents for detection of the at least two targets, which set is
automatically selected(i) wherein one of the reagents in the set is
redundant to another reagent in the set, and the reagents comprise at
least two layers for detection of each of the two targets;(ii) wherein
the sample is contacted with the set of detection reagents;(iii) wherein
the presence or absence of signals from the association of the sets of
detection reagents with the targets is detected; and(iv) wherein the
presence or absence of the signals is correlated with the presence or
absence of targets in the sample.

2. A method comprising automatic selection of a set of reagents to detect
one or more targets in a sample, wherein the set of reagents comprises at
least two layers for detection of each target, and wherein the set
comprises at least one reagent that is redundant to another reagent in
the set.

3. The method of claim 2, wherein at least two reagents in the set are
redundant to other reagents in the set.

4. The method of claim 2, wherein the set of reagents detects two or more
targets in a sample.

5. The method of claim 4, wherein the detection of the first and/or the
second target involves three or more layers of detection reagents.

6. The method of claim 2, wherein at the one or more targets are chosen
from protein targets, DNA targets, and RNA targets.

7. The method of claim 2, wherein information regarding redundancy of the
reagents and information correlating the reagents with the one or more
targets is provided in a computer-generated code.

12. A method comprising automatic selection of a set of reagents to detect
one or more targets in a sample, wherein the set of reagents comprises at
least two layers for detection of each target, wherein the set comprises
at least one reagent that is redundant to another reagent in the set, and
further wherein the automatic selection of the set of reagents comprises
determining, for each reagent: the target or targets the reagent may be
used to detect; the layer of the reagent in each target detection method;
the function of the reagent in the detection method; other reagents in
the set to which the reagent is redundant; and other reagents in the set
with which the reagent will specifically interact.

13. The method of claim 12, wherein, for each reagent in the set,
information regarding: the target or targets the reagent may be used to
detect; the layer of the reagent in each target detection method; the
function of the reagent in the detection method; other reagents to which
the reagent is redundant; and other reagents with which the reagent will
specifically interact, is provided in a computer-generated code.

15. The method of claim 12, wherein the automatic selection is conducted
with the assistance of a computer program.

16. The method of claim 12, wherein the set of reagents detects two or
more targets in a sample.

17. The method of claim 16, wherein the detection of the first and/or the
second target involves three or more layers of detection reagents.

18. The method of claim 12, wherein at least one reagent in the set is
degenerate and comprises a degenerate molecular code.

19. The method of claim 12, wherein the targets are chosen from protein
targets, DNA targets, and RNA targets.

20. A method of detecting one or more targets in a sample, comprising:(a)
obtaining a sample potentially comprising one or more targets;(b)
automatically selecting a set of reagents for detection of the one or
more targets,(i) wherein one of the reagents in the set is redundant to
another reagent in the set, and the reagents comprise at least two layers
for detection of each target;(c) contacting the sample with the set of
detection reagents;(d) detecting the presence or absence of signals from
the association of the sets of detection reagents with the one or more
targets; and(e) correlating the presence or absence of the signals with
the presence or absence of each target in the sample.

21. The method of claim 20, wherein at least two reagents in the set are
redundant to other reagents in the set.

22. The method of 20, wherein the set of reagents detects at least two
targets in a sample.

23. The method of claim 20, wherein the detection of the one or more
targets involves three or more layers of detection reagents for at least
one target.

24. The method of 20, wherein the set of reagents is selected by a method
comprising determining, for each reagent in the set:the target or targets
the reagent may be used to detect; the layer of the reagent in each
target detection method; the function of the reagent in the detection
method; other reagents to which the reagent is redundant; and other
reagents with which the reagent will specifically interact.

25. The method of claim 20, wherein the automatic selection is conducted
with the assistance of a computer program.

Description:

[0002]The instant invention relates to systems of reagents and methods for
the detection of chemical and biological targets in a sample.

[0003]Some embodiments comprise methods for automatically selecting a set
of reagents to detect one or more targets in a sample, wherein the set of
reagents comprises at least two layers for detection of a first target,
and at least two layers for detection of an optional second target,
wherein the set optionally comprises reagents that are at least partially
redundant. In some embodiments, the redundancy is created by at least one
degenerate reagent such that the reagent may interact specifically with
more than one other component of a detection system or sample. In some
embodiments, the system or method also includes reagent containers with a
computer-generated code which may further serve to match targets to
appropriate reagents.

BACKGROUND AND SUMMARY OF THE INVENTION

[0004]Diagnostic or detection assays commonly used in biology and
chemistry, such as Western, Northern, or Southern blots,
immunohistochemistry (IHC), immunocytochemistry, in situ hybridization
(ISH), ELISA, and the like, all operate on the basic principle that a
target in a sample is detected by contacting the target with a probe
which it specifically recognizes, which leads to a detectable change in
the sample that registers as a signal. For instance, the probe may be
linked, either directly or indirectly, to a detectable label, such as a
fluorophore, chromophore, or an enzymatic or radioactive tag, which
provides the signal. Polymeric detection reagents and conjugates are also
compatible with this invention.

[0005]Some detection systems also provide ways of enhancing the signal
from the target. For example, the label's signal may be enhanced by
increasing the number of detectable labels used to detect each target, or
by instrumentation that may amplify the signal. If the target is an
antigen, a multiple-antibody system may amplify the detection signal. For
instance, the target may first be bound by a primary antibody probe,
which, in turn, is capable of binding many secondary antibodies, which,
in turn, may optionally be recognized by tertiary antibodies, which act
as amplification layers. The detectable label, then, may be present on or
recognized by the outermost amplification layer. This method, thus,
increases the strength of the signal from the detection of each target,
as many detectable labels become associated with each target rather than
only one or a few.

[0006]In some cases, one may also wish to detect more than one target in a
sample, either in separate procedures, or simultaneously on the same
portion of the sample. For example, an experimenter may wish to test a
biological sample for the presence of several different genetic targets
or protein targets in order to assist in a diagnostic procedure. It may
be more efficient, in some settings, to perform those assays
simultaneously or immediately following one another on the same part of
the sample, so that the number of steps involved is minimized.

[0007]The present invention allows for a closed reagent system for
detecting one target or more than one target in a sample. The set of
reagents is automatically selected, which may help to ensure accuracy.
For example, the system may choose a correct set of reagents in
pre-optimized amounts and in the correct order, thus reducing human
errors in carrying out the protocols. In some embodiments, the system may
also choose the reaction conditions, and be programmed with information
concerning cross-reactivities of various reagents. Accordingly, the
reagent sets, systems, and methods of the present invention may lessen
the risk of errors in diagnosis due to false positives or false negatives
in a diagnostic assay, by allowing for greater uniformity of application.

[0008]The instant invention includes a system and method for automatically
selecting a set of reagents for a detection protocol, which set may be
used to detect one or more than one target in a sample. The selected set
of reagents comprises at least two layers of reagents for each target to
be detected, and includes at least one redundant reagent. The redundant
reagent, for example, can be replaced by at least one other detection
reagent in the set. Hence, a detection assay, run with the selected
reagent set, can be optimized to choose the appropriate reagent from
among the redundant reagents in the set. In some embodiments, the set of
reagents also includes at least one degenerate reagent, interacts with
more than one other reagent in the set, making that reagent redundant. In
some embodiments, the degenerate reagent, because of its flexibility of
interactions, could be used in the detection of more than one target.

[0009]Such redundancies and reagent interchangeabilities may increase the
efficiency of some detection assays, as they allow for mixing and
matching between different reagents. When degenerate reagents are also
used, that interchangeability may allow for fewer detection reagents in a
particular system, so that multi-target detection is simpler to carry out
and is associated with less risk of unwanted interactions between
detection reagents. Redundancies may also allow a researcher to choose
from more than one type detectable label. That mixing and matching may
also expand the uses of a particular set of detection reagents, so that a
system can be more easily adapted to detecting several different targets,
depending upon the user's needs. For instance, if a particular detectable
label recognizes more than one probe, then that label could be used in
more than one detection assay, thus reducing the number of labels needed
for a given set of assays.

[0010]In some systems of the invention, the degenerate reagent contains at
least one degenerate molecular code such that the same site of the
reagent is capable of specifically binding to more than one other
molecule. In some embodiments, degenerate nucleic acid hybridization may
be used to create a degenerate molecular code. In other embodiments, an
epitope of an antigen may specifically recognize more than one antibody,
for example.

[0011]Alternatively, degeneracy in a reagent could be created with
reagents that contain more than one binding site, each for a different
binding partner. For example, if nucleic acid hybridization is used for
the detection reagents to interact, a degenerate reagent may contain two
different recognition sequences. If antigen-antibody interactions are
used, one may design an antigen such that it has more than one different
epitope. Yet further, molecular entities can be constructed using
chemical linkers and polymers such that they bridge two different binding
elements together in one molecule.

[0012]To aid in automated selection of the reagent set, simple algorithms
may be used to create a set of information about each reagent, such as
its function in the detection method, which targets it is used to detect,
what other detection reagents it interacts with, any redundancies or
degeneracies it has, and at what stage and concentration it is applied to
the sample. In some embodiments, the automated selection of the reagent
set occurs fully or in part through a computer-generated code on the
reagent containers. The computer-generated code may be used to retain the
above information so that when a user desires to detect a particular
target, the correct set of reagents is selected and organized, and
redundant reagents are noted. Examples include bar codes and sku codes,
but other known software-readable signals would suffice. Such automated
reagent selection is compatible with various known automated or
semi-automated detection apparatuses. Further, in some cases, the
automated reagent selection may also allow for certain parts of the
detection procedure on the sample to be carried out manually.

[0013]The instant methods and resulting reagent systems are compatible
with a large variety of samples and are adaptable to a large number of
targets, probes, and detectable labels. For instance, the present
invention is useful in immunohistochemistry applications (IHC) and in
situ hybridization (ISH), and can be applied to other detection methods
as well. Other detection that may be compatible with this invention
include, for example, immunocytochemistry (ICC), flow cytometry, enzyme
immuno-assays (EIA), enzyme linked immuno-assays (ELISA), blotting
methods (e.g. Western, Southern, and Northern), labeling inside
electrophoresis systems or on surfaces or arrays, and precipitation,
among others.

[0014]Such detection formats, for example, are useful in research as well
as in diagnosing diseases or conditions. Further, if multiple targets are
detected, such systems may be useful in analyzing expression patterns of
genes or levels of proteins within a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 illustrates an exemplary two or three detection reagent
system which is compatible with the invention. The probe in this example
is connected to a detectable label by hybridization of nucleic acid
analog segments, one or more of which may comprise a degenerate molecular
code. Both the probe and label are each present on larger molecular
entities. An adaptor unit, shown in the middle panel is optional, but
may, in some cases, serve to link the probe and detectable label through
an intermediate set of nucleic acid hybridizations, and may also serve as
an amplification layer. The nucleic acid analog segments may be present
on one or more of the molecular entities in the system.

[0016]FIG. 2 illustrates an exemplary system and method compatible with
the invention in which a target antigen bound to a primary antibody is
recognized by a recognition unit comprising a secondary antibody probe.
The recognition unit is specifically hybridized to a detection unit via
the nucleic acid analog segments on each unit. In such a system, either
the detection unit or the recognition unit molecular entities may
comprise a degenerate molecular code. The system shown in this figure has
3 layers of detection reagents above the target: the primary antibody,
recognition unit with probe, and detection unit with detectable label.

[0017]FIG. 3 illustrates an exemplary three or higher-layer detection
system and method compatible with the invention wherein a target antigen
bound to a primary antibody is recognized by a recognition unit
comprising a secondary antibody probe and a nucleic acid analog segment.
The recognition unit specifically hybridizes to an adaptor unit
comprising nucleic acid analog segments that specifically hybridize to
the recognition unit and a detection unit. Hence, there are four layers
in this system above the target: the primary antibody, secondary antibody
probe molecular entity, adaptor, and detection unit with detectable
label. Any of the detection reagents may comprise degenerate molecular
codes in their nucleic acid analog segments, for example, or may be
replaceable with a redundant reagent.

[0018]FIGS. 4-6 illustrate exemplary non-natural bases and base-pairings
which may be used in the instant invention to produce nucleic acid-based
degenerate molecular codes in detection reagents.

[0019]FIG. 7 illustrates an exemplary system in which an antigen is
degenerate and is recognized by more than one specific antibody. For
instance, the antigen may incorporate more than one epitope, or an
epitope that is recognized by more than one different antibody.
Alternatively, the same epitope could be bound specifically by different
antibodies. In this example, each different antibody carries a different
detectable label, leading to redundancy, as either one of the antibodies
could be selected for a detection experiment. That redundancy allows for
a choice among detection labels.

[0020]FIG. 8 presents two illustrations showing, first, how a set of
reagents may be include one redundant reagent. In the top illustration,
the same target may be detected by either A, B, and C, or by A, X, and C.
X and B are redundant as each can take the place of the other. The second
panel illustrates how two targets may be detected by overlapping sets of
reagents as one reagent is degenerate. Target 1 is detected by A, B, and
C, while Target 2 is detected by P, B, and D. Reagent B is able to
interact with all of P, A, C, and D, due to a degeneracy in its
recognition properties. Thus, reagent B is degenerate. Reagents C and D
are redundant in that B could interact with either of them. If reagents C
and D are detectable labels, that redundancy allows the experimenter
detecting the targets to select the more appropriate label for the
experiment.

[0021]FIG. 9 illustrates an example of redundancy of detection reagents in
the systems and methods according to some embodiments of the invention.
In FIG. 9, the molecular entity carrying the detectable label may be used
in the detection of Target 1 by binding directly to the molecular entity
carrying the probe. In the detection of Targets 2 and 3, that same entity
binds to an adaptor unit. Thus, the molecular entity is redundant in that
it can be used in more than one detection assay in a system. The adaptor
unit shown in FIG. 8 is also redundant in that it can be used in the
detection of both Targets 2 and 3.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0022]Redundant or redundancy, as applied to the set of reagents herein,
applies to a molecule or reagent that can be replaced with another
reagent in the same set.

[0023]Degenerate, as used herein, applies to a molecule that is able to
specifically bind to more than one other molecule in a set of reagents. A
degenerate molecular code, herein, is one way to produce degeneracy in a
detection reagent. The term applies to a code at the molecular structure
level that recognizes more than one other molecular structure.

[0024]A computer-generated code, as used herein, includes any code that
may be created or interpreted by computer hardware and/or software,
including but not limited to numerical, color, and letter codes.

[0025]Automated or automatic, and the like, refer to non-manual methods.

[0026]Set of as used herein means two or more of any item.

[0027]Detection reagent as used herein means a reagent that is used to
detect a target in a sample by either directly recognizing the target or
by directly recognizing another detection reagent that, in turn, directly
recognizes the target.

[0028]Sample, as used herein, refers to any composition potentially
containing a target.

[0029]Target, as used herein, refers to any substance present in a sample
that is capable of detection.

[0030]The term recognize, and similar terms, when applied to a target or
detection reagent herein, means to render the target detectable by a
detectable label. Recognition includes, for example, reacting with a
target, directly binding to a target, and indirectly reacting with or
binding to a target.

[0031]The terms bind, binding, and similar terms, when applied to the
instant targets and detection reagents, mean an event in which one
substance physically interacts with another. Specific, specific for, or
specifically and similar terms are used to indicate that the binding
between two or more molecular entities is through specific interactions
rather than through non-specific aggregation, for example. Specific
hybridization and like terms as used herein refer to the specific binding
of two single-stranded nucleic acid segments to create double-stranded
nucleic acids.

[0032]Amplify, amplification, and similar terms, mean an increase in the
observed intensity of a signal from a detectable label.

[0033]A protein herein is used in the broadest possible sense, and
includes any molecule comprising a sequence of amino acids, such as a
short peptide, peptide hormone, or protein fragment, and larger molecules
including antibodies, enzymes, glycoproteins, lipoproteins, etc.

[0034]Antibody, as used herein, means an immunoglobulin or a fragment
thereof, and encompasses any polypeptide comprising an antigen-binding
site regardless of the source, method of production, and other
characteristics.

[0035]An antigen, as used herein, refers to any substance recognized by an
antibody.

[0036]As used herein, a nucleic acid, nucleic acid sequence, or nucleic
acid segment is defined in the broadest possible sense and includes a
variety of natural nucleic acids as well as nucleic acid analogs. For
instance, nucleic acid may be any nucleobase sequence comprising any
oligomer, polymer, or polymer segment, wherein an oligomer means a
sequence of two or more backbone monomer units. Backbones include any
substance capable of forming an oligomer, including DNA, RNA, PNA, LNA,
and any modified or substituted backbone. Nucleobases (or bases) may be,
for example, natural bases such as adenine (A), cytosine (C), guanine
(G), thymine (T), and uracil (U), as well as any non-natural base.

[0037]As used herein, the terms base and nucleobase refer to any
purine-like or pyrimidine-like molecule that may be comprised in a
nucleic acid segment or nucleic acid analog segment.

[0041]As used herein, all numbers are approximate, and may be varied to
account for errors in measurement and rounding of significant digits.

Detection Reagents

[0042]Example detection reagents compatible with this invention include
reagents such as probes, which specifically bind to a target in a sample,
or detectable labels, which create a signal which can be detected, to
indicate the presence and/or concentration of a target, and various
reagents that link probes and detectable labels together, such as
molecular adaptors, which physically connect the probe and detectable
label. Adaptors may in some cases serve to amplify a detection signal,
for instance if many adaptor molecules bind to each probe. An example of
such an amplifying adaptor is a secondary antibody, which recognizes the
constant region of a primary antibody probe, such that many secondary
antibodies bind to each primary antibody in the sample. If a detectable
label recognizes the secondary antibody, for instance, an enzyme with a
colored substrate, then many such labels will become associated with each
probe-target in the sample.

[0043]Any or all of the detection reagents described herein may be present
in isolation, or may form part of larger molecular entities, for
instance. One example system of larger, interacting molecular entities is
shown in FIGS. 1-3, in which a probe and detectable label are each
present on an entities that interact through nucleic acid hybridization.
The nucleic acid segments of the molecular entities may be attached to
the probe and detectable label, for instance, via chemical linkers and
polymers. There may also be multiple nucleic acid segments, or more than
one nucleic acid segment on at least one of the molecular entities, such
that the entity is able to interact with more than one specific binding
partner, depending upon the experimental protocol. Alternatively, FIG. 7
illustrates how antigen-antibody interactions can physically connect
different probe-label combinations together.

Automated Reagent Selection and Redundant Reagents

[0044]The instant invention allows for the automatic selection of a set of
reagents to detect one or more targets in a sample, such as for
diagnostic purposes. In such methods, an algorithm may be used to
organize the detection reagents into layers (i.e. probes, adaptors or
amplifiers, and detectable labels) of at least 2 or at least 3. The
algorithm may also specify the targets the reagents are compatible with.
The algorithm may further specify the other detection reagents that each
reagent selected interacts with, including intended interactions to
produce a signal, and any unwanted interactions that might interfere with
labeling. Those methods may, in some embodiments, select a set of
reagents in which the set includes at least two layers of reagents to
detect a first target, and at least two layers of reagents to detect an
optional second target, wherein at least one reagent in the set is
redundant. The redundant reagent may used to replace another reagent in
the set, if needed, or it may not be used in the detection protocol.

[0045]In some embodiments, at least one of the reagents in the set is a
degenerate reagent, and thus, is able to interact with more than one
other reagent in the set. Degenerate reagents may allow other reagents in
the set to be redundant, as the degenerate reagents can interact with
more than one other reagent. FIGS. 7-9, described above, provide
illustrations of redundancies and degeneracies according to the
invention.

[0046]The automated method may be put into action by a computer and
associated software, for example, so that when a user selects a
particular target to identify, the detection system is able to identify
an appropriate set of reagents. When a user selects more than one target,
in some embodiments, the system would be able to select complementary
reagents, including redundant reagents.

[0047]For example, if using a computer and software to select the
reagents, each reagent could be coded based upon a specific set of
parameters, such as, first, its level in the detection method. A probe,
for example, may be at level 1, as it is intended to interact with a
target. Nevertheless, if a probe indirectly interacts with a target
through another entity, or if a blocking step is employed first in the
reaction, a probe could be assigned to a higher level in the
organization, with the blocker or other reagent taking the first level.
An adaptor, if used, could be at level 2, if it directly interacts with
the probe, and a detectable label may be at level 2 or 3 or higher,
depending upon whether an adaptor is used, for example.

[0048]The next parameter for tracking a detection reagent may be which
target(s) or detection scheme(s) it is used for. A given reagent, if
degenerate, may be used in more than one scheme, such as in the detection
of more than one target. (See FIG. 8, lower panel.) Or it may be used
only in one scheme. Further, a computer could select the appropriate
reagent for an assay from among those that are redundant and hence,
interchangeable.

[0049]A reagent may accordingly be assigned to a particular target or
target panel, to track whether it is used in a test for targets A, B,
and/or C, for example. A reagent may also be assigned a further parameter
that relates to its function, separate from its level in the overall
system. For example, a reagent may be assigned a function as a probe, but
could be, as explained above, at level 1 or 2, depending upon the
organization of the detection assay.

[0050]By using at least one reagent in a set that is redundant, or
interchangeable, a particular detection scheme could be automatically
modified to choose from more than one set of detection reagents for a
given target. This might help to avoid, for example, unwanted
interactions between two parallel target detection schemes run on the
same sample. A reagent could be substituted with its redundant reagent,
in addition, if its stock is running low.

[0051]Accordingly, the present application also allows for a method for
automatic selection of a set of reagents to detect one or more targets in
a sample, wherein the set of reagents comprises at least two layers for
detection of a first target and at least two layers for detection of a
second target, wherein the set comprises at least one redundant reagent.

[0052]In addition, the computer-generated code may include other
information about the detection reagent. Examples include the reagent's
reaction conditions with the sample (i.e. incubation times and
temperatures), any unwanted cross-reactivities it has, the strength of
the signal it produces, whether a washing or blocking step should be
performed in conjunction, etc.

Degenerate Reagents and Degenerate Molecular Codes

[0053]In some embodiments, at least one member of a reagent set is
degenerate such that it can interact with more than one other molecule in
the set. Degeneracies may be created by designing the detection reagents
to contain two different binding sites. For example, an antigen may
contain more than one epitope or a segment of nucleic acid may contain
more than one protein binding site or nucleic acid hybridization site.

[0054]In some embodiments, one binding site may be itself degenerate, and
thus capable of interacting with more than one binding partner. Such a
degeneracy may be formed a degenerate molecular code. One example of such
a code is a nucleic acid segment or sequence that is capable of specific
hybridization to more than one other nucleic acid segment or sequence.
Such nucleic acid codes may be generated, for instance, from the use of
non-natural bases that form stable base-pairing interactions with more
than one other natural or non-natural base. Nucleic acid codes and their
associated binding rules may additionally be input into an optional
computer or software program, such that the program can determine from
the sequences of the nucleic acids what other detection reagents the
sequence should interact with.

[0055]Non-natural bases that could be used to make a degenerate molecular
code may include, for example, purine-like and pyrimidine-like molecules,
such as those that may interact using Watson-Crick-type, wobble, or
Hoogsteen-type pairing interactions. Examples include generally any
nucleobase referred to elsewhere as "non-natural" or as an "analog."

[0057]Yet other examples include bases in which one amino group with a
hydrogen is substituted with a halogen (small "h" below), such as
2-amino-6-"h"-purines, 6-amino-2-"h"-purines, 6-oxo-2-"h"-purines,
2-oxo-4-"h"-pyrimidines, 2-oxo-6-"h"-purines, 4-oxo-2-"h"-pyrimidines.
Those will form two hydrogen bond base pairs with non-thiolated and
thiolated bases;

[0058]For example, some specific embodiments of non-natural bases are the
structures shown in FIG. 4 with the following substituents, which are
described in the PCT Application entitled "New Nucleic Acid Base Pairs,"
which is incorporated herein by reference.

[0059]In other examples, one or more of the H or CH3 are
independently substituted with a halogen such as Cl or F. Other example
non-natural bases and base-pairs are shown in FIG. 20 herein. R1 in the
structures of FIGS. 4-6 may serve as a point of attachment to a backbone
group, such as PNA, DNA, RNA, etc. Still other examples are illustrated
in FIGS. 2(A) and 2(B) of Buchardt et al. (U.S. Pat. No. 6,357,163).

[0060]Non-natural bases such as those exemplified above may be able to
form stable base pairing interactions with more than one other base, via
2 or 3-hydrogen bond schemes, for example. The chart below and figures
provided herein illustrate several examples of how such degenerate
base-pairing schemes lead to the ability to synthesize nucleic acid
analog segments with degenerate recognition.

[0061]In some embodiments, the degenerate molecular codes are nucleic acid
analog segments made from DNA or RNA backbones and at least one
non-natural base. In other embodiments, they are made from nucleic acids
of non-natural backbone units as well. Such non-natural backbone units
thus include, but are not limited to, for example, PNA's, LNA's or
phosphorothioate or 2'O-methyl nucleosides.

[0062]For example, in some embodiments, one or more phosphate oxygens may
be replaced by another molecule, such as sulfur. In other embodiments, a
different sugar or a sugar analog may be used, for example, one in which
a sugar oxygen is replaced by hydrogen or an amine, or an O-methyl. In
yet other embodiments, nucleic acid analog segments comprise synthetic
molecules that can bind to a nucleic acid or nucleic acid analog. For
example, a nucleic acid analog may be comprised of peptide nucleic acids
(PNAs), locked nucleic acids (LNAs), or any derivatized form of a nucleic
acid. Such backbone units may be attached to any base, including the
natural bases A, C, G, T, and U, and non-natural bases.

[0063]As used herein, "peptide nucleic acid" or "PNA" means any oligomer
or polymer comprising at least one or more PNA subunits (residues),
including, but not limited to, any of the oligomer or polymer segments
referred to or claimed as peptide nucleic acids in U.S. Pat. Nos.
5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336,
5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610,
5,986,053, 6,107,470 6,201,103, 6,228,982 and 6,357,163; all of which are
herein incorporated by reference.

[0065]As used herein, the term "locked nucleic acid" or "LNA" means an
oligomer or polymer comprising at least one or more LNA subunits. As used
herein, the term "LNA subunit" means a ribonucleotide containing a
methylene bridge that connects the 2'-oxygen of the ribose with the
4'-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).

[0067]Different nucleic acid analog segments may hybridize, for instance,
using Watson-Crick-type, wobble, or Hoogsteen-type base-pairing.
Accordingly, the nucleic acid analog segments comprise sequences which
allow for hybridization to take place at a desired stringency.

[0068]In other embodiments of the invention, degenerate molecular codes
may be created from other types of molecular interactions. For instance,
a given antigen epitope or a given substrate may be recognized by more
than one antibody or enzyme. (See, for example, FIG. 7.)

Systems and Methods for Detection

[0069]Some embodiments of this invention are based on the principle of
using the automatically selected reagent sets as part of a system of
detection reagents for one or more targets. Such redundant, and
optionally, degenerate detection reagents may, for instance, allow for a
degree of interchangeability from one detection protocol to another. (See
FIGS. 8-9.) For instance, in some embodiments of the invention, a
molecular entity carrying a detectable label specifically interacts with
a probe or with another molecular entity comprising the probe.

[0070]For example, if the probe is degenerate, that probe could in turn
interact with more than one type of detectable label. Such interactions
would then allow the experimenter to choose a suitable label for the
experiment from among those the probe specifically recognizes, allowing
for greater flexibility. Accordingly, the two interchangeable detectable
labels become redundant reagents in the system. In other cases, should
supplies of one type of label run low, a different adaptor or label could
be substituted without adversely affecting the detection assay.

[0071]In other examples, a degenerate probe could specifically bind to
more than one type of adaptor molecular entity, allowing the adaptors to
be redundant. If the different adaptors interact with different
detectable labels or molecular entities carrying those labels, then the
method of labeling could similarly be expanded. In other embodiments, the
detectable label or molecular entity carrying the label could be
degenerate, such that it could be used to bind specifically to more than
one type of probe or adaptor. In yet other embodiments, the adaptor could
be degenerate, allowing it to link together more than one
probe-detectable label combination. (An example is shown in the bottom
panel of FIG. 8.)

[0072]The present invention also contemplates a system for detecting one
or more targets in a sample, comprising at least one set of detection
reagents for each target, each set comprising at least two detection
reagents, wherein at least one detection reagent in the system is
degenerate, and optionally comprises a degenerate molecular code, and
wherein, optionally, each container for each detection reagent comprises
a computer-generated code, wherein the degenerate molecular code and the
computer-generated code allow each set of detection reagents to associate
with its intended target.

[0073]In some systems, the degenerate reagents could be made from nucleic
acids, such as a nucleic acid analog segment that specifically hybridize
to more than one other nucleic acid segment, or two different nucleic
acid segments, each with different binding properties. In some
embodiments, the nucleic acid is PNA or LNA rather than DNA or RNA. (See,
e.g., FIGS. 4-6.) In other systems, the degenerate reagent comprises at
least one hapten or at least one antigen. For example, an antigen may be
specifically recognized by more than one antibody, either because it
contains one epitope with multiple binding partners or because it
contains two or more different epitopes. (See, e.g., FIG. 7.) Such
systems may be used to detect, for example, protein targets, DNA targets,
and RNA targets in a sample, as well as other molecules or entities such
as carbohydrates, membrane lipids, chemical toxins, and the like.

[0074]Such systems may be used to in manual or automated detection assay
protocols. The computer-generated codes may be helpful in assigning and
moving reagents in an automated process, for example. In either case, the
systems may be used within detection apparatuses. Such apparatuses may,
for instance, serve to dispense reagents in appropriate amounts, apply
washing steps, hold the samples, incubate the samples at appropriate
temperatures during a detection process, and detect signal.

[0075]Some apparatuses may employ computer hardware and software to
control the progress of the detection method, such as the dispensing of
reagents. For instance, computer software could be used to select the set
of reagents, and also to move and dispense reagent containers for a
particular detection method at the appropriate time. In such instances, a
computer-generated code on the containers may be helpful in controlling
the apparatus.

[0076]In some systems, the computer-generated code comprises a bar code,
or another type of known coding for tracking the use or movement of
containers, such as a sku code. Even simple numbering systems, colors, or
other computer-detectable codes may suffice. In some embodiments, the
computer-generated code could include information about the function and
level of the detection reagents and their interactions with each other,
as well as information such as how long and at what temperature they
should be incubated with the sample, what activity or signal levels they
have, what unwanted cross-reactivities they show, and whether blocking or
washing steps should be performed.

[0077]The instant invention also involves detection methods, for instance,
A method of detecting one or more targets in a sample, comprising

(a) obtaining a sample potentially comprising one or more targets;(b)
automatically selecting a set of reagents for detection of the one or
more targets,

[0078](i) wherein one of the reagents in the set is redundant to another
reagent in the set, and the reagents comprise at least two layers for
detection of each of the two targets;

(c) contacting the sample with the set of detection reagents;(d) detecting
the presence or absence of signals from the association of the sets of
detection reagents with the one or more targets; and(e) correlating the
presence or absence of the signals with the presence or absence of the
one or more targets in the sample.

[0079]In some embodiments, at least one reagent in the set is degenerate.
For instance, that reagent may contain a degenerate molecular code, such
as a nucleic acid code comprising at least one non-natural nucleic acid
base, as described above. In others, the degeneracy could be formed from
other types of interactions, such as antigen-antibody interactions or
other protein-ligand interactions.

[0080]In some embodiments, two or more targets are detected.

[0081]In some embodiments, two or more targets are detected. The reagent
sets and methods of the invention may be used to detect, for example,
protein targets, DNA targets, and RNA targets in a sample, as well as
other molecules or entities such as carbohydrates, membrane lipids,
chemical toxins, and the like.

[0082]In some methods, a computer-generated code is used for the automated
selection of the reagent set. That code may, for instance, help to
determine whether the redundant reagent should be substituted in the
detection assay for its interchangeable partner. The computer-generated
code may also help to organize the dispensing and control of the various
reagents by their properties, such as what layer or function in the
detection scheme they have, which target detection they belong to, what
redundancies and/or degeneracies they have, etc.

[0083]In some detection methods, the level of the target in the sample may
be detected qualitatively. For instance, in some cases the experimenter
is interested only in the presence or absence of a target. However, in
other cases, the experimenter may wish to detect the target
quantitatively, to also determine its relative concentration or amount in
the sample. In such cases, methods such as densitometry could be employed
to convert the signal from a target into a quantitative or digital
reading. If a computer-associated apparatus is used, the apparatus and
software could be adapted to quantitatively read the signal generated
from the detection method. Alternatively, a separate densitometry
apparatus and program could be employed.

Automated Selection of Staining Schemes and Reagent Sets

[0084]The automated selection of a reagent set may optionally also include
optimizing various parameters such as the overall time of the reaction or
the signal intensity that results. The user may also prefer certain
stains over other available choices. The available quantity of a given
reagent may also be a factor to consider. Hence, a computer-generated
code may include information related to those user preferences so that a
given software algorithm may select an optimized set of reagents. The
coded data can be both general for the particular reagent and lot
dependant, for example including:

[0085]Specific binding pattern with different species

[0086]Unwanted cross reactivity with different species

[0087]Blocking reagents

[0088]Reaction kinetics at different temperatures and dilutions

[0089]Amplification power

[0090]Activity as function of age

as well as more traditional data, such as:

[0091]Reagent Name

[0092]Lot number

[0093]Production date

[0094]Storage conditions

[0095]Expiry

[0096]Volume.

[0097]Multi-parameter coding may also assist in ensuring the consistency
and reproducibility of a diagnostic assay, such as one performed
according to an approved regulatory protocol for diagnosing disease. In
some embodiments, it may thus be helpful to effectively "lock" the system
so that the user must allow automated selection of the reagents and
optionally, the detection protocol. Further, when coding multiple
parameters, only a computer may be able to interpret the information in a
reasonable manner. In other embodiments, the system may be "open" or
"partially open" such that the general user is aware of how the set of
reagents is being selected and can amend the selection procedure if
needed.

WORKING EXAMPLES

Example 1

Coded Detection Reagents

[0098]Assume a goat anti-mouse HRP polymeric conjugate gives a 6-fold
amplification compared to a monomeric conjugate, at reaches saturation
after 10 min incubation at 20° C. or after 4 minutes at 25°
C. Further assume that 4-fold dilution of the antibody reagent has the
same effect as doubling the incubation time. Further assume that the
reagent containing goat IgG, has an HRP enzyme with moderate enzyme
activity, is specific against mouse IgG, has 2% cross reactivity against
rabbit IgG, 15% cross reactivity against rat IgG, contains BSA, gives 5%
background after 2 wash cycles and 2% background after 3 wash cycles, and
that the reagent's enzymatic activity drops by 5% each month after the
production date.

[0099]That information could be represented in the form of a code such as:
6-10.20.4.25-4.2-G-HM-M-2R-15Ra-B-5.2.2.3-5.

[0100]A similar code could be designed to incorporate information about
interacting nucleic acid segments as well. An example could be a similar
polymeric reagent containing alkaline phosphatase (AP) and two binding
entities with sequence TCD-DGsGs-TAC-A and CAT-DGsD-ATC-Gs. Those binding
entities could be made from DNA, or a non-natural backbone such as PNA,
for example. The binding pattern would be specific against
UsGUs-DPP-TTG-D and PGD-UsTP-TDUs-G, respectively, for example.

[0101]If the reagent gives 4-fold amplification compared to a monomeric
conjugate; reaches saturation after 8 min at 20° C. and after 2
minutes after 25° C.; if 5-fold dilution of the reagent has the
same effect as doubling the incubation time; the reagent contains AP
enzyme with a high enzyme activity; contains BSA, gives 4% background
after 2 wash cycles and 2% after 3 wash cycles; and if the activity drops
by 3% per months from production date, then a code to represent the
reagent could be as follows:
4-8.20.2.25-5.2-TCDDGsGsTACA-CATDGsDATCGs-AH-B-4.2.2.3-3.

[0102]In the code above, each number or letter represents a piece of
information about the reagent, as provided above.

Example 2

Exemplary Reagent Systems

[0103]An example of a reagent system based on conventional reagents in a
ready to use (RTU) format may be as follows:

[0108]Polymeric conjugates give about 5-10 times stronger signals than
conventional reagents. Thus, those reagents require half the incubation
time of conventional reagents. A DAB staining of the ER protein could be
done by one of the following five protocols below, each giving
approximately the same staining results. The letters represent the
addition of the successive reagents above and the incubation time after
each addition.

[0109]Optionally, temperature variables may also be included. The
protocols do not include optional washing and blocking steps, for
simplicity.

[0110]The protocols may start with the same primary reagent and same
chromogen, but use different intervening reagents, and hence, different
steps. Protocol #2 have the fewest steps and is the longest, whereas
protocol #5 uses more steps and has a shorter incubation time. Reagent E
is used in step 3, while D is used in step 2 of the protocol,
respectively. If both visualization reagents were not available in the
system due to a low volume or due to instrument scheduler constraints,
protocol #2 and #3 could still be performed. The software could
accordingly help the user to select the best overall staining scheme,
given the time constraints and available reagents. Each reagent above may
further be coded as described in Example 1.

Example 3

Additional Exemplary Reagent Systems

[0111]A reagent system based on reagents in a ready to use (RTU) format is
as follows:

[0121]Note: CAT-DGsD-ATC-Gs ("Erna") specifically hybridizes to
PGD-UsTP-TDUs-G ("Elmer"), while TCD-DGsGs-TAC-A ("Anna") specifically
hybridizes to UsGUs-DPP-TTG-D ("Alex"). The sequence AAA-AAA-AAA
specifically hybridizes to UsUsUs-TTT and TTT-UsUsUs, whereas UsUsUs-TTT
and TTT-UsUsUs do not bind to each other.

[0122]A DAB staining of the ER protein is carried out using one of the
protocols below, which, for simplicity, do not include optional washing
and endogen peroxidase blocking steps. The steps below list the reagent
to be added and the incubation time. Coding to change or control
temperature may also be included.

[0126]Accordingly, a set of reagents in the system depicted in this
example allows for multiple types of staining protocols, because some of
the reagents have degenerate binding patterns.

Example 4

Preparation and Use of Detection Reagents and Systems Using Molecular
Entities Interacting Through Nucleic Acid Base-Pairs

Example 4a

Preparation of Pyrimidinone-Monomer

[0127]1. In dry equipment 4.6 g of solid Na in small pieces was added to
400 mL ethanol (99.9%), and was dissolved by stirring. Hydroxypyrimidine
hydrochloride, 13.2 g, was added and the mixture refluxed for 10 minutes.
Then 12.2 mL ethyl-bromoacetate (98%) was added and the mixture refluxed
for 11/2 hour. The reaction was followed using Thin Layer Chromatography
(TLC). The ethanol was evaporated leaving a white compound, which was
dissolved in a mixture of 80 mL of 1M NaCitrate (pH 4.5) and 40 mL of 2M
NaOH. This solution was extracted four times with 100 mL Dichloromethane
(DCM). The DCM phases were pooled and washed with 10 mL NaCitrate/NaOH--
mixture. The washed DCM phases were evaporated under reduced pressure and
resulted in 17.2 g of crude solid product. This crude solid product was
recrystallized with ethylacetate giving a yellow powder. The yield for
this step was 11.45 g (63%).

[0128]2. The yellow powder, 12.45 g. from above was hydrolyzed by
refluxing overnight in a mixture of 36 mL DIPEA, 72 mL water and 72 mL
dioxane. The solvent was evaporated and water was removed from the
residue by evaporation from toluene. The yield for this step was 100%.

[0129]3. OBS. Pyrimidinone acetic acid (10.5 g), 16.8 g PNA-backbone
ethylester, 12.3 g DHBT-OH, 19 mL Triethylamine was dissolved in 50 mL
N,N-dimethylformamide (DMF). DIPIDIC (11.8 mL) was added and the mixture
stirred overnight at room temperature. The product was taken up in 100 mL
DCM and extracted three times with 100 mL of dilute aqueous NaHCO3.
The organic phase was extracted twice with a mixture of 80 mL of 1M
NaCitrate and 20 mL of 4M HCl. Because TLC showed that some material was
in the citrate phase, it was extracted twice with DCM. The organic phases
were pooled and evaporated. Because there was a precipitation of urea,
the product was dissolved in a DCM, and the urea filtered off. Subsequent
evaporation left an orange oil. Purification of the orange oil was
performed on a silica column with 10% methanol in DCM. The fractions were
collected and evaporated giving a yellow foam. The yield for this step
was 7.0 g (26.8%).

[0130]4. The yellow foam (8.0 g) was hydrolyzed by reflux overnight in 11
mL DIPEA, 22 mL water, and 22 mL dioxane. The solvent was evaporated and
the oil was dehydrated by evaporation from toluene leaving an orange
foam. The yield for this step was 100%.

Example 4b

Second Method of Preparing Pyrimidonone Monomer

[0131]Step 1. In dry equipment 9.2 g of solid Na in small pieces was
dissolved in 400 mL ethanol (99.9%), with stirring. Hydroxypyrimidine
hydrochloride, 26.5 g, was added, and the mixture was stirred for 10
minutes at 50° C. Then 24.4 mL Ethyl bromoacetate (98%) was added
and the mixture stirred at 50° C. for 1 hour. The reaction was
followed using Thin Layer Chromatography (TLC).

[0132]The ethanol was evaporated leaving a white compound, which was
dissolved in 70 mL of water and extracted with 20 mL DCM. Another 30 mL
of water was added to the water phase, which was extracted with
3×100 mL DCM. The DCM-phase from the first extraction contains a
lot of product, but also some impurities, wherefore this phase was
extracted twice with water. These two water phases then were back
extracted with DCM.

[0133]The combined DCM phases were pooled and washed with 10 mL water. The
washed DCM phases were evaporation under reduced pressure and resulted in
25.1 g yellow powder. The yield for this step was 25.1 g=69%. Maldi-Tof:
181.7 (calc. 182).

[0134]Step 2. 34.86 g yellow powder from above was dissolved in 144 mL 2M
NaOH. After stirring 10 minutes at room temperature, the mixture was
cooled in an ice bath. Now 72 mL 4 M HCl (cold) was added. The product
precipitated. After stirring for 5 minutes, the precipitate was filtered
and thoroughly washed with ice water. Drying in a dessicator under
reduced pressure left 18.98 g yellow powder. The yield for this step was
18.98 g=64%.

[0136]The product was taken up in 100 mL DCM and extracted with
2×100 mL dilute aqueous NaHCO3. Both of the aqueous phases were
washed with a little DCM. The organic phases were pooled and evaporated.
Evaporation left an orange oil. Purification of the product was done on a
silica column with 10-20% methanol in ethylacetate. The fractions were
collected and evaporated giving a yellow oil. The oil was dissolved and
evaporated twice from ethanol. The yield from this step was 20.68 g=90%.

[0137]Step 4. The yellow oil (18.75 g) was dissolved in 368 mL 0.2 M
Ba(OH)2. Stirring for 10 minutes before 333 mL 0.221 M H2SO4 was
added. A precipitation was performed immediately. Filtration through
cellite, which was washed with water. The solvent was evaporated. Before
the evaporation was at end, the product was centrifuged to get rid of the
very rest of the precipitation. Re-evaporation of the solvent left a
yellow oil. The yield from this step was 13.56 g=78%.

[0138]Step 5. To make a test on the P-monomer 3 consecutive P's were
coupled to Boc-L300-Lys(Fmoc) resin, following normal PNA standard
procedure. The product was cleaved from the resin and precipitated also
following standard procedures: HPPP-L300-Lys(Fmoc). Maldi-Tof on the
crude product: 6000 (calc. 6000) showing only minor impurities.

Example 4c

Preparation of the Thio-Guanine Monomer

[0139]1. 6-Chloroguanine (4.93 g) and 10.05 g K2CO3 was stirred
with 40 mL DMF for 10 minutes at room temperature. The reaction mixture
was placed in a water bath at room temperature and 3.55 mL ethyl
bromoacetate was added. The mixture was stirred in a water bath until TLC
(20% Methanol/DCM) showed that the reaction was finished. The
precipitated carbonate was filtered off and washed twice with 10 mL DMF.
The solution, which was a little cloudy, was added to 300 ml water,
whereby it became clear. On an ice bath the target compound slowly
precipitated. After filtration the crystals were washed with cold ethanol
and dried in a desiccator. The yield for this step was 3.3 g (44.3%) of
ethyl chloroguanine acetate.

[0140]2. Ethyl chloroguanine acetate (3.3 g) was dissolved by reflux in 50
mL absolute ethanol. Thiourea (1.08 g) was added. After a refluxing for a
short time, precipitate slowly began forming. According to TLC (20%
Methanol/DCM) the reaction was finished in 45 minutes. Upon completion,
the mixture was cooled on an ice bath. The precipitate was then filtered
and dried overnight in a desiccator. The yield for this step was 2.0 g
(60%) ethyl thioguanine acetate.

[0141]3. Ethyl thioguanine acetate (3.57 g) was dissolved in 42 mL DMF.
Benzylbromide (2.46 mL) was then added and the mixture stirred in an oil
bath at 45° C. The reaction was followed using TLC (25%
Methanol/DCM). After 3 hours all basis material was consumed. The step 3
target compound precipitated upon evaporation under reduced pressure and
high temperature. The precipitate was recrystallized in absolute ethanol,
filtered and then dried in a desiccator. The yield for this step was 3.88
g (82%) of methyl benzyl thioguanine ethylester.

[0142]4. Methyl benzyl thioguanine ethylester (5.68 g) was dissolved in
12.4 mL of 2M NaOH and 40 mL THF, and then stirred for 10 minutes. The
THF was evaporated by. This was repeated. The material was dissolved in
water and then 6.2 mL of 4M HCl was added, whereby the target product
precipitated. Filtering and drying in a desiccator. The yield for this
step was 4.02 g (77%).

[0143]5. The product of step 4 (4.02 g), 3.45 g backbone ethylester, 9 mL
DMF, 3 mL pyridine, 2.1 mL triethylamine and 7.28 g PyBop were mixed and
then stirred at room temperature. After 90 minutes a solid precipitation
formed. The product was taken up in 125 mL DCM and 25 mL methanol. This
solution was then extracted, first with a mixture of 80 mL of 1M
NaCitrate and 20 mL of 4M HCl, and then with 100 mL dilute aqueous
NaHCO3. Evaporation of the organic phase gave a solid material. The
material was dissolved in 175 mL boiling ethanol. The volume of the
solution was reduced to about 100 mL by boiling. Upon cooling in an ice
bath, the target product precipitate. The crystals were filtered, washed
with cold ethanol and then dried in a desiccator. The yield of this step
was 6.0 g (86%.)

[0144]6. The product of step 5 (6.0 g) was dissolved in 80 mL THF, 7.5 mL
2M NaOH and 25 mL water. The solution became clear after ten minutes of
stirring. THF was evaporated. Water (50 mL) was added to the mixture. THF
was evaporated. Water (50 mL) was added to the mixture. When the pH was
adjusted by the addition of 3.75 mL of 4M HCl, thio-guanine monomer
precipitated. It was then filtered, washed with water and dried in a
desiccator. The yield for this step was 5.15 g (91%).

Example 4d

Preparation of Diaminopurine Acetic Acid Ethyl Ester

[0145]1. Diaminopurine (10 g) and 40 g of K2CO3 were added to 85
mL of DMF and stirred for 30 minutes. The mixture was cooled in a water
bath to 15° C. Ethyl bromoacetate (3 mL) was added three times
with 20 minute intervals between each addition. This mixture was then
stirred for 20 minutes at 15° C. The mixture was left in the water
bath for another 75 minutes, and the temperature increased to 18°
C. The DMF was removed by filtering and the remaining K2CO3 was
added to 100 mL of ethanol and refluxed for 5 minutes. Filtering and
repeated reflux of the K2CO3 in 50 mL ethanol, filtering. The
pooled ethanol phases were placed in a freezer, after which crystals
formed. These crystals were filtered, washed with cold ethanol, filtered
again and then dried in a desiccator overnight. The yield for this step
was 12 g (76%).

Example 4e

Preparation of an L30-Linker to Connect Elements of a Multicomponent
Molecular Entity

[0146]This linker is one example of a linking element that may covalently
bridge elements of a polymeric molecular entity. Multiple L30 linker
segments may be strung together to create a longer linker, if desired.

[0147]1. A solution of 146 mL of 2,2'-(Ethylenedioxy)bis(ethylamine) (98%)
in 360 mL of THF was cooled in an ice bath. Di-tert-butyl dicarbonate
(97%) (65 g) in 260 mL THF was added dropwise over one hour. The solvent
was evaporated. The remaining oil was dissolved in water and then
evaporated off. The oily product was dissolved in 300 mL water, extracted
with 300 mL DCM, then washed twice with 150 mL of DCM. The collected
organic phase was washed with 50 mL of water before evaporating to about
half the volume. The organic phase was then extracted with 400 mL of 1M
NaCitrate (pH 4.5), and then extracted again with 50 mL of 1M NaCitrate
(pH 4.5). The aqueous phases were washed with 50 mL DCM before cooling on
an ice bath. While stirring, 100 mL of 10M NaOH was added to the aqueous
washed aqueous phases resulting in pH of 13-14. In a separation funnel
the product separated on its own. It was shaken with 300 mL DCM and 50 ml
water. The organic phase was evaporated, yielding a white oil. The yield
for this step was 48.9 g (65.7%). The product had a predicted molecular
formula of C11H24N2O4 (MW 248.3).

[0148]2. Boc-amine (76.2 g) was dissolved in 155 mL pyridine. Diglycolic
anhydride (54.0 g) (90%) was added. After stirring for 15 minutes the
intermediate product separated out and then 117 mL Acetic Anhydride (min.
98%) was added and the mixture stirred at 95° C. for 1 hour. The
solution was then put under reduced pressure and evaporated. Water (117
mL) was added, and the mixture was then stirred for 15 minutes, after
which 272 mL of water and 193 mL of DCM were added. The organic layer was
extracted twice with 193 mL of 1M Na2CO3 and then twice with a
mixture of 72 mL of 4M HCl and 289 mL of 1M NaCitrate. After each
extraction the aqueous phase was washed with a little DCM. The collected
organic phase was washed with 150 mL of water. The solvent was evaporated
leaving the product as an orange oil. This yield for this step was 100.3
g (0.29 mol) (94%). The product had a predicted molecular formula of
C15H26N2O7 (MW 346.4).

[0149]3. The product from step 2 (100.3 g) was dissolved in an equal
amount of THF and was then added dropwise to 169.4 mL of
2,2'-(Ethylendioxy)bis(ethylamine) at 60° C. over the period of 1
hour. The amine was distilled from the reaction mixture at 75-80°
C. and a pressure of 3×10-1 mBar. The residue from the
distillation was taken up in a mixture of 88 mL of 4M HCl and 350 mL of
1M NaCitrate and then extracted three times with 175 mL of DCM. The
aqueous phase was cooled in an ice bath and was cautiously added to 105
mL of 10M NaOH while stirring. In a separation funnel the product slowly
separated from the solution. When separated 100 mL of water and 950 mL of
DCM were added to the product. Stirring for some minutes before pouring
to a separation funnel. The pH in the aqueous phase should be 14. The
aqueous phase was extracted four times with 150 mL of DCM. The solvent
was evaporated. The oily residue was dehydrated by evaporation from
toluene, giving a yellow oil. The yield for this step was 115.48 g (81%).
The product had a predicted molecular formula of
C21H42N4O9 (MW 494.6).

[0150]4. The Boc-amine (115.48 g) from step 3 was dissolved in 115 mL of
pyridine. Diglycolic anhydride (40.6 g) (90%) was added and the mixture
stirred for 15 minutes, after which the intermediate product came out.
Acetic Anhydride (97 mL) (min. 98%) was added and the mixture stirred at
95° C. for 1 hour. The mixture was then evaporated under reduced
pressure. The mixture was then cooled and then 80 mL of water was added.
This mixture was stirred for 15 minutes and then 200 mL of water and 150
mL of DCM were added. The organic layer was extracted twice with 150 mL
of 1M Na2CO3 and then twice with a mixture of 53 mL of 4M HCl
and 213 mL of 1M NaCitrate. After each extraction the aqueous phase was
washed with a little DCM. The collected organic phase was washed with 150
mL of water. The solvent was evaporated. The oily residue was dehydrated
by evaporation from toluene, giving a yellow oil. The yield for this step
was 125 g (92%). The product had a predicted molecular formula of
C25H44N4O12 (MW 592.6), with a mass spectrometry
determined molecular weight of 492.5.

[0151]Further purifying of the product could be done on a silica column
with a gradient from 5-10% methanol in DCM. The yield from the column
purification was 69% and produced a white oil.

[0152]5. White oil (12.4 g) from step 4 was dissolved in a mixture of 12
mL water and 12 mL 1,4-Dioxane (99%) and was then heated to reflux. DIPEA
(6 mL) was added and refluxed for 30 minutes. This mixture was cooled and
then evaporated. The oily residue was dehydrated by evaporation from
toluene, giving a yellow oil. The product had a predicted molecular
formula of C25H46N4O14 (MW 610.6).

[0155]Using standard procedures provided below, an MBHA-resin was loaded
with Boc-Lys(Dde)-OH. Using a peptide synthesizer, amino acids were
coupled according to PNA solid phase procedure provided in Example 18d
yielding Boc-L90-Lys(Fmoc)-L30-Lys(Dde). The Boc and Fmoc
protections groups were removed and the amino groups marked with
flourescein using the procedure in Example 18e. Then, the Dde protection
group was removed and 0.4 M cysteine was added according to the procedure
in Example 18b. The PNA was cleaved from the resin, precipitated with
ether and purified on HPLC according to Example 18d. The product was
found to have a molecular weight of 3062 using MALDI-TOF mass
spectrometry; the calculated molecular weight is 3061.

Example 4i

Synthesis of a Conjugate Made from Sequence AA from Example 5, DexVS70,
and Flu(10)

[0156]Dextran (with a molecular weight of 70 kDa) was activated with
divinylsulfone to a degree of 92 reactive groups/dextran polymer; this
product is designated DexVS70.

[0157]The above four compounds were mixed. The mixture was placed in a
water bath at 30° C. for 16 hours. The mixture was added to 50
nmol of freeze-dried PNA (sequence AA from Example above). The mixture
was placed in a water bath at 30° C. for 30 minutes. The
conjugating reaction was quenched with 50 μL of 500 mM cysteine for 30
minutes at 30° C. Purification of the product was performed using
FPLC: column SUPERDEX®--200, buffer 10 mM Hepes 100 mM NaCl, method 7
bank 2, Loop 1 mL. Two fractions were collected: one with the product and
one with the residue. The relative absorbance Flu2
(ε.sub.500nm=146000 M-1, ε260nm=43350 M-1)
and PNA (ε.sub.500nm=73000 M-1, ε260nm=104000
M-1) was used to calculate the average conjugation ratio of
Flu2, PNA, and DexVS70. The conjugation ratio of Flu2 to
DexVS70 was 9.4. The conjugation ratio of PNA (sequence AA) to DexVS70
was 1.2.

Example 4j

Synthesis of HRP-DexVS70-Seq. AA

[0158]Using the procedure of Example 4o below, the conjugate
HRP-DexVS70-Seq. AA was made. The ratio of HRP to DexVS70 is 12.2; the
ratio of Seq. AA to Dex70 is 1.2.

Example 4k

Synthesis of GaM-DexVS70-Seq. AB

[0159]The synthesis of GaM-DexVS70-Seq. AB was performed using the
procedure in Example 16 with the following changes as indicated.

[0161]The above five components were mixed and placed in a water bath at
30° C. for 40 minutes. Two hundred and ninety μL were taken out
of the mixture and added to 100 nmol of Seq. AB, which was previously
dissolved in 80 μL of H2O. Then, 20 μL of 0.8 M NaHCO3
(pH 9.5) was added and the mixture placed in a water bath at 30°
C. for 1 hour. Quenching was performed by adding 39 μL of 500 mM
cysteine and letting the resultant mixture set for 30 minutes at
30° C.

[0162]Purification of the product on FPLC: column SUPERDEX®--200,
buffer 10 mM Hepes 100 mM NaCl, method 7 bank 2, Loop 1 mL. Two fractions
were collected: one with the product and one with the residue. Relative
absorbance PNA(Flu) (ε.sub.500nm=73000 M-1) and GAM
(ε278nm=213000 M-1) (correction factor for PNA at 278
nm is due to the specific PNA and is calculated: 278/500 nm) was used to
calculate the average conjugation ratio of PNA, GAM and DexVS70. The
ratio of PNA to DexVS70 was 5.3 and the ratio of GaM to DexVS70 was 0.8.

[0165]The above four compounds were mixed and placed in a water bath at
30° C. for 65 minutes. From this mixture, 875 μL was taken out
and added to the indicated number of nmol of PNA in the table below;
before the addition the PNA had been dissolved in the μL of H2O
indicated in the table below. Then the number of μLs of 0.8 M
NaHCO3 (pH 9.5) was added according to the table below. The
resulting mixture was placed in a water bath at 30° C. for 70
minutes. Quenching was performed by adding 6 mg of solid cysteine (0.05
M) to the mixture and letting it stand for 30 minutes at 30° C.

[0166]Purification of the product on FPLC: column SUPERDEX®--200,
buffer 10 mM Hepes 100 mM NaCl, method 7 bank 2, Loop 1 mL. Two fractions
were collected: one with the product and one with the residue. Relative
absorbance PNA(Flu) (ε.sub.500nm=73000 M-1) and AHB
(Pε278nm=213000 M-1) (correction factor for PNA at 278
nm is due to the specific PNA and is calculated: 278/500 nm) was used to
calculate the average conjugation ratio of PNA, AHB and DexVS70.

[0167]Conjugates with different ratios PNA are shown in the following
table.

[0169]1. An MBHA-resin was loaded with
Boc-L30-Lys(Fmoc)-L90-Lys(Fmoc)-L90-Lys(Fmoc) using a
standard loading procedure to a loading of 0.084 mmol/g.

[0170]2. To this resin, Boc-Lys(Fmoc)-L30-AAC-GGG-ATA-ACT-GCA-CCT-was
coupled using the peptide synthesizer machine following standard PNA
solid phase chemistry. Fmoc protection groups were removed and the amino
groups were labeled with fluorescein. After cleaving and precipitation
the PNA was dissolved in TFA. The precipitate was washed with ether. The
precipitate was dissolved in 200 μL NMP To this solution 6 mg Fmoc-Osu
was added and dissolved. Next, DIPEA (9 μL) was added and the reaction
was followed using MALDI-TOF mass spectrometry. After 30 minutes the
reaction was finished and the PNA was precipitated and washed with ether.

[0171]HPLC after dissolving the PNA in 30% CH3CN and 10% TFA/H2O
gave three pure fractions. The fractions were pooled and lyophilized. The
lyophilized PNA was then dissolved in 192 μL NMP. Piperidine (4 μL)
and 4 μL DBU was added to this solution which set for 30 minutes.
Analysis by MALDI-TOF mass spectrometry gave a molecular weight of 10777.

[0172]The precipitate was washed with ether and was then dissolved in 100
μL TFA. The precipitate was washed with ether and then dried using
N2 gas.

[0174]The above five components are mixed together placed in a water bath
at 30° C. for 16 hours. Five hundred microliters of this mixture
are added to 50 nmol PNA, which is previously dissolved in 40 μL
H2O. Then, 10 μL of 0.8 M NaHCO3 (pH 9.5) is added. The
mixture is then placed in a water bath at 30° C. for 2 hours.
Quenching is performed by adding 55 μL of 110 mM cysteine and letting
the resultant mixture set for 30 minutes at 30° C.

[0176]Two fractions are collected: one with the product and one with the
residue. Relative absorbance HRP (ε404nm=83000 M-1,
ε.sub.500nm=9630 M-1) and PNA(Flu)
(ε.sub.500nm=73000 M-1) is used to calculate the average
conjugation ratio of HRP, PNA and DexVS70.

[0178]The above five components are mixed and placed in a water bath at
30° C. for 40 minutes. Two hundred and ninety μL is taken out
of the mixture and added to 50 nmol of PNA, which is previously dissolved
in 40 μL of H2O. Then, 10 μL of 0.8 M NaHCO3 (pH 9.5) is
added and the mixture placed in a water bath at 30° C. for 1 hour.
Quenching is performed by adding 34 μL of 500 mM cysteine and letting
the resultant mixture set for 30 minutes at 30° C.

[0179]Purification of the product on FPLC: column SUPERDEX®--200,
buffer 10 mM Hepes 100 mM NaCl, method 7 bank 2, Loop 1 mL. Two fractions
are collected: one with the product and one with the residue. Relative
absorbance PNA(Flu) (ε.sub.500nm=73000 M-1) and GAM
(ε278nm=213000 M-1) (correction factor for PNA at 278
nm is due to the specific PNA and is calculated: 278/500 nm) was used to
calculate the average conjugation ratio of PNA, GAM and DexVS70.

Example 4a

Standard Synthesis of PNA1-DexVS70-PNA2

[0180]Dextran (molecular weight 70 kDa) is activated with divinylsulfone
to a degree of 92 reactive groups/dextran polymer. PNA1 (100 nmol) is
dissolved in 140 μL of DexVS70 (10 nmol). To this mixture 12.5 μL
of PNA2 (12.5 nmol) dissolved in H2O is added, and then 30 μL of
NaHCO3 (pH 9.5) is added and the solution mixed. The resultant
mixture is placed in a water bath at 30° C. for 35 minutes.
Quenching was performed by adding 18.3 μL of 500 mM cysteine in Hepes
and letting this mixture set for 30 minutes at 30° C.

[0181]Purification of the product on FPLC: column SUPERDEX®--200,
buffer 10 mM Hepes 100 mM NaCl, method 7 bank 2, Loop 1 mL. Two fractions
are collected: one with the product and one with the residue. Relative
absorbance PNA(Flu) (ε.sub.500nm=73000 M-1) and the
proportion between the two PNA's is used to calculate the average
conjugation ratio of PNA, PNA and DexVS70.

[0188]The Boc-PNA-2-Thiouracil-(S-4-MeOBz)-monomer was prepared according
to Jesper Lohse, Otto Dahl and Peter E. Nielsen; Proceedings of the
National Academy of Science of the United States of America, 1999, Vol
96, Issue 21, 11804-11808.

[0193]IsoAdenine (2-aminopurine) may be prepared as a PNA-monomer by 9-N
alkylation with methylbromoacetate, protection of the amino group with
benzylchloroformate, hydrolysis of the methyl ester, carbodiimide mediate
coupling to methyl-(2-Boc-aminoethyl)-glycinate, and finally hydrolysis
of the methyl ester.

[0194]4-thiouracil may be prepared as a PNA-monomer by S-protection with
4-methoxy-benzylchloride, 1-N alkylation with methylbromoacetate,
hydrolysis of the methyl ester, carbodiimide mediate coupling to
methyl-(2-Boc-aminoethyl)-glycinate, and finally hydrolysis of the methyl
ester.

[0195]Thiocytosine may be prepared as a PNA monomer by treating the
Boc-PNA-cytosine(Z)-monomer methyl ester with Lawessons reagent, followed
by hydrolysis of the methyl ester.

[0196]A number of halogenated bases are commercially available, and may be
converted to PNA monomers analogously to the non-halogenated bases. These
include the guanine analog 8-bromo-guanine, the adenine analogs
8-bromo-adenine and 2-fluoro-adenine, the isoadenine analog
2-amino-6-chloro-purine, the 4-thiouracil analog 5-fluoro-4-thio-uracil,
and the 2-thiouracil analog 5-chloro-2-thiouracil.

[0197]Boc-PNA-Uracil monomers were first described in "Uracil og
5-bromouracil I PNA," a bachelor project by Kristine Kilsa Jensen,
Kobenhavns Universitet 1992.

Example 4s

Miscellaneous Standard Procedures

[0198]a. Loading of resins. P-methyl-BHA-resin (3 g) is loaded with
Boc-Lys(Fmoc)-OH 15 mmol/g resin. The lysine is dissolved in NMP and
activated with 0.95 equivalents (eq.) HATU and 2 eq. DIPEA. After loading
the resin, it is capped by adding a solution of (Ac)2O/NMP/pyridine
(at a ratio of 1/2/2) and letting it set for at least 1 hour or until
Kaiser test was negative. After washing with DCM, the resin is dried in a
dessicator. Quantitative Kaiser test typically gives a loading of 0.084
mmol/g.

[0199]b. Amino Acid Couplings. The Boc protection group is removed from
the resin with TFA/m-cresol (at a ratio of 95/5) 2×5 min. The resin
is then washed with DCM, pyridine and DMF before coupling with the amino
acid, which is dissolved in NMP in a concentration between 0.2 and 0.4 M
and activated with 0.95 eq. of HATU and 2 eq of DIPEA for 2 minutes. The
coupling is complete when the Kaiser test is negative. Capping occurring
by exposing the resin for 3 minutes to (Ac)2O/pyridine/NMP (at a
ratio of 1/2/2). The resin is then washed with DMF and DCM

[0200]c. Boc-L300-Lys(Fmoc)-resin. To the loaded Boc-Lys(Fmoc)-resin,
L30-Linker in a concentration of 0.26 M was coupled using standard
amino acid coupling procedure. This was done 10 times giving
Boc-L300-Lys(Fmoc)-resin.

[0201]d. PNA solid phase. On a peptide synthesizer (ABI 433A, Applied
Biosystems) PNA monomers are coupled to the resin using standard
procedures for amino acid coupling and standard PNA chemistry. Then the
resin is handled in a glass vial to remove protections groups and to
label with either other amino acids or fluorophores.

[0202]Removal of the indicated protection groups is achieved with the
following conditions:

[0203]Boc: TFA/m-cresol (at a ratio of 95/5) 2×5 min.

[0204]Fmoc: 20% piperidine in DMF 2×5 min.

[0205]Dde: 3% hydrazine in DMF 2×5 min.

[0206]When the synthesis is finished, the PNA is cleaved from the resin
with TFA/TFMSA/m-cresol/thioanisol (at a ratio of 6/2/1/1). The PNA is
then precipitated with ether and purified on HPLC. MALDI-TOF mass
spectrometry is used to determine the molecular weight of the product.

[0207]e. Labeling with fluorescein. 5(6)-carboxy fluorescein is dissolved
in NMP to a concentration of 0.2 M. Activation is performed with 0.9 eq.
HATU and 1 eq. DIPEA for 2 min before coupling for at least 2×20
min or until the Kaiser test is negative.

Example 4t

PNA with Positive and Negative Loadings

[0208]In order to make better conjugations at one time we tried to give
the PNA a loading. Both PNA's were made by PNA standard procedures (See
Example 4s above).

[0209]1. Flu-L30-Glu-TCA-AGG-TAC-A-Glu-L300-Lys(Cys)

[0210]Glu=glutamate has negative loadings and for the easiness the PNA is
designated -A4-

[0211]2. Flu-L30-Lys(Me)2-TGT-ACC-TTG-A-Lys(Me)2-L330--
Lys(cys)

[0212]Lys(Me)2=Boc-Lys(Me)2-OH has positive loadings and the PNA
is designated +T+

[0214]The tissue samples were placed in a marked plastic histocapsule
(Sakura, Japan). Dehydration was performed by sequential incubation in
70% ethanol twice for 45 min, 96% ethanol twice for 45 min, 99% ethanol
twice for 45 min, and xylene twice for 45 min. The samples were
subsequently transferred to melted paraffin (melting point 56-58°
C.) (Merck, Whitehouse Station, N.J.) and incubated overnight (12-16
hours) at 60° C. The paraffin-infiltrated samples were transferred
to fresh warm paraffin and incubated for an additional 60 min prior to
paraffin embedding in a cast (Sekura, Japan). The samples were cooled to
form the final paraffin blocks. The marked paraffin blocks containing the
embedded tissue samples were stored at room temperature in the dark.

3. Cutting, Mounting and Deparaffination of Embedded Samples

[0215]The paraffin blocks were cut and optionally also mounted in a
microtome (0355 model RM2065, Feather S35 knives, set at 5.0 micrometer;
Leica, Bannockburn, Ill.). The first few millimeters were cut and
discarded. Paraffin sections 4-6 micrometers thick were then cut and
collected at room temperature. The sections were gently stretched on a
45-60° C. hot water bath before being mounted onto marked
microscope glass slides (SUPERFROST® Plus; Fisher, Medford, Mass.),
two tissue sections per slide. The slides were then dried and baked in an
oven at 60° C. The slides were deparaffinated by incubating twice
in xylene for 5 min±2 min twice, then in 96% ethanol for 2 min+/-30
sec, then twice in 70% ethanol for 2 min+/-30 sec, and then once in
Tris-buffered saline with TWEEN® (called herein TBST) for 5 min. TBST
comprises 50 mM Tris adjusted to pH 7.6 with HCl; 150 mM NaCl; 0.05%
TWEEN®20. The slides were deparaffinated by subsequently incubation
in xylene twice for 5 min±2 min, 96% ethanol twice for 2 min+/-30 sec
and 70% ethanol twice for 2 min+/-30 sec. The slides were immersed in
deionized water and left for 1 to 5 min.

4. Endogenous Peroxidase Blocking

[0216]Samples were incubated with a 3% hydrogen peroxide solution for 5
min. to quench endogenous peroxidase activity, followed by washing in
deionized water for 1 to 5 min.

5. Antigen Retrieval by Microwave Oven

[0217]Antigens in the sample were retrieved by immersing the slides in a
container containing Antigen Retrieval Solution, pH 6.0 (DakoCytomation
code No. K5204 Vial 7 or optional code No. K5205 Vial 7). The container
was closed with a perforated lid and placed in the middle of a microwave
oven and left boiling for 10 min. The container was removed from the oven
and allowed to cool at room temperature for 20 min. The samples were
rinsed in deionized water.

6. Antigen Retrieval by Water Bath Incubation

[0218]Antigens in the sample were retrieved by immersing the slides in a
beaker containing Antigen Retrieval Solution, pH 6.0 (DakoCytomation code
No. K5204 Vial 7 or optional code No. K5205 Vial 7). The samples were
incubated for 40 min in a water bath at 95-100° C. The beaker was
removed from the water bath and allowed to cool at room temperature for
20 min. The samples were rinsed in deionized water.

7. Water-Repellent Barrier to Liquids by DakoCytomation Pen

[0219]To ensure good coverage of reagent on the tissue sample, the area on
the slide with tissue was encircled with a silicone rubber barrier using
DakoCytomation Pen (DakoCytomation code No. 2002). The slides were
transferred to a rack and placed in a beaker containing Tris-buffered
saline with TWEEN® (called herein TBST) and left for 5 min. TBST
comprises 50 mM Tris adjusted to pH 7.6 with HCl; 150 mM NaCl; 0.05%
TWEEN®20.

8. Application of a Primary Antibody

[0220]Monoclonal Mouse anti-Human Cytokeratin (DakoCytomation code No.
M3515) diluted 1:900 in ChemMate® Antibody Diluent (DakoCytomation
code No. S2022) was applied on the tissue samples and incubated for 30
min in a humid chamber at ambient temperature. The slides were
individually rinsed and then washed in TBST for 5 min.

[0223]In the above tables, the letters A, C, G, U, and T, stand for the
natural bases adenine, cytosine, guanine, uracil, and thymine. P stands
for pyrimidinone, D for 2,6-diaminopurine, and Us for 2-thiouracil.

11. Fixation of PNA1-Conjugate with 1% Glutardialdehyde

[0224]The samples were washed in deionized water for 30 sec. Then, 1%
glutardialdehyde (Merck Art. No. 820603), called herein GA, diluted in 22
mM calcium phosphate buffer, pH 7.2, was applied, and the samples were
incubated for 10 min in a humid chamber at ambient temperature. The
samples were washed in deionized water for 30 sec and in TBST for 5 min.

12. Application of a PNA1-PNA2/Dextran Conjugate Adaptor Unit

[0225]PNA1-PNA2/Dextran conjugate is also called
"PNA1-PNA2" in the following examples. Table 7 summarizes the
compositions of PNA1-PNA2 conjugates. PNA1 is
complementary to the PNA1 conjugate, and PNA2 is complementary to
the PNA2 conjugates D14079 and D13155 described in step 13 below. The
sequence of PNA1 is CUsGsGsDD TUsD GsDC and
the sequence of PNA2 is UsGUs DPP TTG D, in which Us
stands for 2-thio-uracil, Gs stands for 2-amino-6-thioxopurine, D
stands for diaminopurine, and P stands for pyrimidinone. The conjugates,
diluted in BBA, were applied to the tissue samples in a range of
dilutions, and the samples were then incubated for 30 min in a humid
chamber at ambient temperature. The samples were individually rinsed and
washed in TBST for 5 min. When testing a PNA1-PNA2 conjugate,
fixed concentrations of 0.08 μM PNA1 and 0.05 μM PNA2 were used.

[0226]Horse Radish Peroxidase (HRP)/Dextran/PNA2 conjugates are also
called "PNA2 conjugate" in the examples that follow, and are listed in
table 8. The PNA2 conjugates comprise 70.000 Da molecular weight dextran.
The conjugates diluted in BBA were applied to the tissue samples in a
range of dilutions, and samples were incubated for 30 min in a humid
chamber at, ambient temperature. The samples were individually rinsed and
washed twice in TBST for 5 min.

[0227]The diaminobenzidine chromogenic substrate solution, DAB+
(DakoCytomation code No. K3468) was applied on the tissue samples, and
the samples were incubated for 10 min in a humid chamber at ambient
temperature. The samples were washed with deionized water for 5 min.

[0230]The tissue staining was examined in a bright field microscope at
10×, 20× or 40× magnification. Both the specific and
the non-specific staining intensity were described with a score-system
using the range 0 to 3+ with 0.5+ score interval. ChemMate®
EnVision® Detection kit Rabbit/Mouse (DakoCytomation code No. K5007
bottle A) was used as a reference, and was included in all experiments
for testing in parallel with the PNA conjugates. K5007 was used according
to manufacturer's instructions. The antibodies were used in the following
dilutions: M3515 at 1:900, M0755 at 1:8000, and M7240 at 1:1200. The
staining intensity of the K5007 reference using the primary antibody
M3515 diluted 1:900 was set to 2+ in order to compare and assess the
staining result of the PNA conjugate tested. If the reference deviated
more than ±0.5, the test was repeated.

[0231]In the examples, the various visualization system combinations of
the invention were tested on routine tissue samples. The staining
performance was compared with a reference visualization system, using
EnVision® and a very dilute antibody from DakoCytomation. The
practical dynamic range of quantitative IHC may be narrow, and e.g.
strongly stained (+3) tissues are not easy to compare with respect to
intensity. Therefore, on purpose, the staining intensity of the reference
system was adjusted to be approximately +2. This was done in order to
better monitor and compare differences in staining intensity with the
system of the invention.

[0235]The mixture was placed in a water bath at 40° C. for 3 hours.
Quenching was performed by adding 30.6 μL of 0.1M ethanolamine and
letting the mixture stand for 30 minutes in water bath at 40° C.
The product was purified on FPLC with: Column Superdex-200, buffer: 2 mM
HEPES, pH 7.2; 0.1M NaCl; 5 mM MgCl2; 0.1 mM ZnCl2. Two fractions were
collected, one with the product and one with the residue.

[0236]In comparison to the experiment described above, another conjugate
was made with extended conjugation time. The three components below were
mixed together and placed in a water bath at 40° C. for 30
minutes.

[0238]The mixture was placed in a water bath at 40° C. for 5 hours.
Quenching was performed by adding 30.6 μL 0.1M Ethanolamine and
letting the mixture stand for 30 minutes in water bath at 40° C.
Purification of the product on FPLC: Column Superdex-200, buffer: 2 mM
Hepes, pH 7.2; 0.1M NaCl; 5 mM MgCl2; 0.1 mM ZnCl2. Two fractions were
collected: One with the product and one with the residue.

[0239]Relative absorbance PNA(Flu) (ε500 nm=73000M-1) and
AP(ε278 nm=140000M-1. Corrected for absorbance from PNA at 278
nm, this correction factor is due to the specific PNA and it is
calculated: 278/500 nm) was used to calculate the average conjugation
ratio of PNA, AP and DexVS70.

[0240]AP-DexVS70-PNA, 3 hrs:

[0241]PNA/DexVS70:1.8

[0242]AP/DexVS70:1.8

[0243]AP-DexVS70-PNA, 5 hrs:

[0244]PNA/DexVS70: 2.0

[0245]AP/DexVS70: 2.4

[0246]Due to these results, it is recommended to follow a procedure in
which the conjugation time (AP+DexVS70-PNA) is 5 hours.

[0248]Goat-anti-mouse secondary antibody conjugated with dextran and a
first PNA sequence (GaM-dex-PNA1 (218-117)) was diluted to final
concentration of 0.08 μM (based on dextran) in BP-HEPES-buffer (1.5%
BSA, 3% PEG, 0.15M NaCl, 10 mM HEPES, pH 7.2) and was applied to the
section. Following 10 minutes incubation at room temperature (RT), the
section was washed 5 minutes using 10× diluted S3006 (Dako). The
sections were rinsed in deionized water. Following 10 minutes incubation
in 0.5% glutaraldehyde at RT, the sections were rinsed in deionized water
and washed 5 minutes using 10× diluted S3006 (Dako).

[0249]An adaptor unit comprising dextran coupled to two different PNA
sequences, one complementary to PNA1 above (PNA2) and another not
complementary to PNA1 (PNA3), called PNA2-dex-PNA3 (218-057) was diluted
to a final concentration of 0.05 μM (dextran) in BP-HEPES-buffer (1.5%
BSA, 3% PEG, 0.15M NaCl, 10 mM HEPES, pH 7.2) and was applied to the
section. Following 10 minutes incubation at RT, the section was washed 5
minutes using 10× diluted S3006 (Dako). Next, a conjugate of a
PNA4, complementary to PNA3 above, dextran, and the detectable label
alkaline phosphatase (PNA4-dex-AP (209-177)) was diluted to final
concentration of 0.05 μM (dextran) in BAP-HEPES-buffer (1.5% BSA, 3%
PEG, 0.15M NaCl, 0.05% 4-aminoantipyrin, 10 mM HEPES, pH 7.2), and was
applied. Following minutes incubation at RT, the sections were washed 5
minutes using 10× diluted S3006 (Dako).

[0251]Primary rabbit antibody A0452 (Dako) targeting CD3 was diluted to
final 1:100 in S2022 (Dako) and applied on a multi tissue section.
Following 10 minutes incubation at RT the sections were washed 5 minutes
using 10× diluted S3006 (Dako).

[0252]Then, a goat-anti-rabbit secondary antibody coupled to dextran and a
first PNA sequence, PNA2a (GaR-dex-Alexander (209-127)) was diluted to
final concentration of 0.08 μM (dextran) in BP-HEPES-buffer (1.5% BSA,
3% PEG, 0.15M NaCl, 10 mM HEPES, pH 7.2) and was applied. Following 10
minutes incubation at RT the sections were washed 5 minutes using
10× diluted S3006 (Dako). The sections were rinsed in deionized
water. Following 10 minutes incubation in 0.5% glutaraldehyde at RT the
sections were rinsed in deionized water and washed 5 minutes using
10× diluted S3006 (Dako).

[0255]General Procedural Note: Before conducting the detection experiment
on formalin-fixed, paraffin-embedded (FPPE) tissue sections, the specimen
should be deparaffinized (dewaxed), rehydrated, and blocked for
endogenous peroxidase activity. Some specimens should be subjected to
target retrieval using heat or enzyme digestion. Following target
retrieval, the specimens should be rinsed gently with wash buffer.

[0256]In this experiment, a mouse primary antibody was used as a primary
binding agent for a specific target in a tissue sample. That antibody was
then recognized by a goat-anti-mouse-dextran-PNA conjugate recognition
unit. A different primary antibody, a rabbit antibody, was used as a
primary binding agent for a different target in the sample. That antibody
was recognized by a goat-anti-rabbit-dextran-PNA recognition unit. One
reaction was visualized by a PNA-dextran-HRP (horse-radish peroxidase)
detection unit and the other reaction was visualized by a PNA-dextran-AP
(alkaline phosphatase) detection unit. PNA sequences 1 and 2 and
sequences 3 and 4, respectively, specifically hybridize to each other.

[0258]The samples were then incubated for 10 minutes in 0.5%
glutaraldehyde at RT and then rinsed in deionized water and washed 5
minutes using 10× diluted S3006 (Dako). PNA2-dex-HRP and
PNA4-dex-AP were both diluted to final concentration of 0.05 M (dextran)
in BAP-HEPES-buffer (1.5% BSA, 3% PEG, 0.15M NaCl, 0.05%
4-aminoantipyrin, 10 mM HEPES, pH 7.2). The two conjugates were applied
simultaneously on the sections. Following 10 minutes incubation at RT the
sections were washed 5 minutes using 10× diluted S3006 (Dako).
Permanent Red working solution (K0640 Dako) and DAB+ working solution (an
aqueous imidazole buffer with hydrogen peroxide and DAB; K3468 Dako) were
prepared.

[0259]The reactions were detected with one of the following methods.
Detection method 1: Permanent Red working solution was applied. Following
10 minutes incubation the sections were washed 5 minutes using 10×
diluted S3006 (Dako). Then DAB+ working solution was applied and
following 10 minutes incubation the sections were washed 5 minutes using
deionized water. Finally the sections were counter stained 5 minutes
using haematoxylin S3301 (Dako), rinsed in deionized water, washed 3
minutes in wash buffer, and mounted in Faramount S3025 (Dako).

[0261]Result: Cytokeratin=HRP=3+ specific staining and 0 background
staining, S100=AP=3+ specific staining and 0 background staining. The
order of detection affects the staining result. If detection method 1 is
used then Permanent Red dominates. If detection method 2 is used then
DAB+ dominates.

[0263]Following 10 minutes incubation at RT the sections were washed 5
minutes using 10× diluted S3006 (Dako). The sections were rinsed in
deionized water. Following 10 minutes incubation in 1% glutaraldehyde at
RT the sections were rinsed in deionized water and washed 5 minutes using
10× diluted S3006 (Dako). PNA2-dex-HRP (209-157) and PNA4-dex-AP
(209-177) were both diluted to final concentration of 0.05 M/dex in
BAP-HEPES-buffer (1.5% BSA, 3% PEG, 0.15M NaCl, 0.05% 4-aminoantipyrin,
10 mM HEPES, pH 7.2). The two conjugates were applied simultaneously on
the sections. Following 10 minutes incubation at RT the sections were
washed 5 minutes using 10× diluted S3006 (Dako). Permanent Red
working solution (K0640 Dako) and DAB+ working solution (K3468 Dako) were
prepared.

[0264]The reactions were detected with one of the following methods.
Detection method 1: Permanent Red working solution was applied. Following
10 minutes incubation the sections were washed 5 minutes using 10×
diluted S3006 (Dako). Then DAB+ working solution was applied and
following 10 minutes incubation the sections were washed 5 minutes using
deionized water. Finally the sections were counter stained 5 minutes
using haematoxylin S3301 (Dako), rinsed in deionzed water, washed 3
minutes in wash buffer, and mounted in Faramount S3025 (Dako).

[0267]In this example, a mouse antibody primary binding agent was
recognized by a GaM-dex-PNA1 and a rabbit antibody primary binding agent
was recognized by GaR-dex-PNA2. One reaction was detected by a
PNA-dex-Enzyme1 conjugate and the other by a PNA-dex-PNA adaptor unit and
then a PNA-dex-Enzyme2 conjugate. PNA1 recognizes PNA2 while PNA3
recognizes PNA4. The enzymes used were HRP and AP, bringing along
respectively a brown and red end-product within the same tissue section.
The PNA-dex-PNA adaptor unit adds a third layer to the detection system.

[0269]GaM-dex-PNA1 (218-117) and GaR-dex-PNA3 (209-127) were both diluted
to final concentration of 0.08 μM (dextran) in BP-HEPES-buffer (1.5%
BSA, 3% PEG, 0.15M NaCl, 10 mM HEPES, pH 7.2). The two conjugates were
applied simultaneously on the sections. Following 10 minutes incubation
at RT the sections were washed 5 minutes using 10× diluted S3006
(Dako). The sections were rinsed in deionized water. Following 10 minutes
incubation in 0.5% glutaraldehyde at RT the sections were rinsed in
deionized water and washed 5 minutes using 10× diluted S3006
(Dako).

[0271]The reactions were detected with one of the following methods.
Detection method 1: Permanent Red working solution was applied. Following
10 minutes incubation the sections were washed 5 minutes using 10×
diluted S3006 (Dako). Then DAB+ working solution was applied and
following 10 minutes incubation the sections were washed 5 minutes using
deionized water. Finally the sections were counter stained 5 minutes
using haematoxylin S3301 (Dako), rinsed in deionized water, washed 3
minutes in wash buffer, and mounted in Faramount S3025 (Dako).

[0275]GaM-dex-PNA1 (218-117) and GaR-dex-PNA3 (209-127) were both diluted
to final concentration of 0.08 M (dextran) in BP-HEPES-buffer (1.5% BSA,
3% PEG, 0.15M NaCl, 10 mM HEPES, pH 7.2). The two conjugates were applied
simultaneously on the sections. Following 10 minutes incubation at RT the
sections were washed 5 minutes using 10× diluted S3006 (Dako). The
sections were rinsed in deionized water. Following 10 minutes incubation
in 0.5% glutaraldehyde at RT the sections were rinsed in deionized water
and washed 5 minutes using 10× diluted S3006 (Dako).

[0278]Permanent Red working solution (K0640 Dako) and DAB+ working
solution (K3468 Dako) were prepared. The reactions were detected with one
of the following methods. Detection method 1: Permanent Red working
solution was applied. Following 10 minutes incubation the sections were
washed 5 minutes using 10× diluted S3006 (Dako). Then DAB+ working
solution was applied and following 10 minutes incubation the sections
were washed 5 minutes using deionized water.

[0281]This example presents a 2-layer detection of two targets in which
mouse-Ab-dex-PNA is recognized by PNA-dex-Enzyme1 and rabbit-Ab-dex-PNA
is recognized by PNA-dex-Enzyme2. The enzymes are HRP and AP bringing
along respectively a brown and red end-product within the same tissue
section. As in preceding examples, PNA1 and 2 specifically hybridize, as
do PNA3 and 4.

[0282]CD3-dex-PNA1 (D16043) and MIB-1-dex-PNA2 (218-097) were both diluted
to final concentration of 0.1 M (dextran) in BP-HEPES-buffer (1.5% BSA,
3% PEG, 0.15M NaCl, 10 mM HEPES, pH 7.2). The two conjugates were applied
simultaneously on the sections. Following 10 minutes incubation at RT the
sections were washed 5 minutes using 10× diluted S3006 (Dako).

[0284]The reactions were detected with one of the following methods.
Detection method 1: Permanent Red working solution was applied. Following
10 minutes incubation the sections were washed 5 minutes using 10×
diluted S3006 (Dako). Then DAB+ working solution was applied and
following 10 minutes incubation the sections were washed 5 minutes using
deionized water. Finally the sections were counter stained 5 minutes
using haematoxylin S3301 (Dako), rinsed in deionized water, washed 3
minutes in wash buffer, and mounted in Faramount S3025 (Dako).

[0293]A method for automatic selection of a set of reagents to detect one
or more targets in a sample, wherein the set of reagents comprises at
least two layers for detection of each target, wherein the set comprises
at least one reagent that is redundant to another reagent in the set.

[0294]The method of Example 5, wherein at least two reagents in the set
are redundant to other reagents in the set.

[0295]The method of Example 5, wherein the set of reagents detects two or
more targets in a sample. The method above, wherein the two or more
targets are detected in the same portion of the sample. The method above,
wherein the two or more targets are detected separately in different
portions of the sample.

[0296]The method of Example 5, wherein the detection of the first and/or
second target involves three or more layers of detection reagents.

[0297]A method for automatic selection of a set of reagents to detect one
or more targets in a sample, wherein the set of reagents comprises at
least two layers for detection of each target, wherein the set comprises
at least one reagent that is redundant to another reagent in the set, and
further wherein the set of reagents is selected by a method comprising
determining, for each reagent in the set:

[0298]the target or targets the reagent may be used to detect;

[0299]the layer of the reagent in each target detection method;

[0300]the function of the reagent in the detection method;

[0301]the other reagents to which the reagent is redundant; and

[0302]the other reagents with which the reagent will specifically
interact.

[0303]The information above could be placed into a computer-generated
code, for example, comprising specific information about each detection
reagent. Optionally, a computer-generated code may also include other
information about the detection reagents such as the incubation
conditions, their cross reactivities with other reagents, and information
concerning adjustments to the reaction protocol that could be made when
using a particular reagent. For instance, the code could note the level
of amplification an amplification reagent achieves, whether a blocking
agent should be used with the particular detection reagent, and the
relative signal strength of a detectable label.

[0304]The method of Example 5, which is conducted with the assistance of a
computer program. The methods above, wherein a computer-generated code on
the containers of the reagents to be selected for the set comprises
information used to select the reagents for the set. The methods above,
wherein the computer-generated code comprises a bar code.

[0305]The methods above, wherein at least one reagent in the set is
degenerate. The methods above, wherein the degenerate reagent comprises a
degenerate molecular code. The methods above, wherein the degenerate
molecular code is a nucleic acid code comprising at least one non-natural
nucleic acid base. The methods above, wherein the nucleic acid code is
comprised within a segment of PNA or LNA. The methods above, wherein the
degenerate molecular code comprises at least one hapten or at least one
antigen.

[0306]The methods above, wherein the at least two targets are chosen from
protein targets, DNA targets, and RNA targets.

Example 6

[0307]A detection apparatus for carrying out any one of the methods above.

Example 7

[0308]A method of detecting one or more targets in a sample, comprising:

(a) obtaining a sample potentially comprising one or more targets;(b)
automatically selecting a set of reagents for detection of the one or
more targets,

[0309](i) wherein one of the reagents in the set is redundant to another
reagent in the set, and the reagents comprise at least two layers for
detection of each target;

(c) contacting the sample with the set of detection reagents;(d) detecting
the presence or absence of signals from the association of the sets of
detection reagents with the one or more targets; and(e) correlating the
presence or absence of the signals with the presence or absence of each
target in the sample.

[0310]The method of Example 7, wherein at least two reagents in the set
are redundant to other reagents in the set.

[0311]The method of Example 7, wherein the set of reagents detects at
least two targets in a sample. The method of Example 7, wherein the set
of reagents detects more than two targets in a sample. The methods above,
wherein the targets are detected in the same portion of the sample. The
methods above, wherein the targets are detected separately in different
portions of the sample.

[0312]The method of Example 7, wherein the detection of the one or more
targets involves three or more layers of detection reagents for at least
one target.

[0313]The method of Example 7, wherein the set of reagents is selected by
a method comprising determining, for each reagent in the set:

[0314]the target or targets the reagent may be used to detect;

[0315]the layer of the reagent in each target detection method;

[0316]the function of the reagent in the detection method;

[0317]the other reagents to which the reagent is redundant; and

[0318]the other reagents with which the reagent will specifically
interact.

[0319]The method of Example 7, which is conducted with the assistance of a
computer program. The methods above, wherein a computer-generated code on
the containers of the reagents to be selected for the set comprises
information used to select the reagents for the set.

[0320]Optionally, a computer-generated code may also include other
information about the detection reagents such as the incubation
conditions, their cross reactivities with other reagents, and information
concerning adjustments to the reaction protocol that could be made when
using a particular reagent. For instance, the code could note the level
of amplification an amplification reagent achieves, whether a blocking
agent should be used with the particular detection reagent, and the
relative signal strength of a detectable label.

[0322]The method of Example 7, wherein at least one reagent in the set is
degenerate. The method above, wherein the degenerate reagent comprises a
degenerate molecular code. A method above, wherein the degenerate
molecular code is a nucleic acid code comprising at least one non-natural
nucleic acid base. The method above, wherein the nucleic acid code is
comprised within a segment of PNA or LNA.

[0323]The method of Example 7, wherein the degenerate molecular code
comprises at least one hapten or at least one antigen.

[0325]A detection apparatus for carrying out any one of the methods
described above for Example 6 and its variants.

Example 9

[0326]A software algorithm for automated selection of a set of detection
reagents according to any one of the methods described in the examples
above.

Sequence CWU
1

23110DNAArtificial SequenceDescription of Artificial Sequence Synthetic
PNA oligonucleotide which does not occur in nature 1tcnnggtaca
10210DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 2catngnatcg
10310DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 3ugunnnttgn
10410DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 4ngnutntnug
10512DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 5cuggnntung nc
12612DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 6gtntaattnn ag
12712DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 7ngtcgnnggu cu
12812DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 8agacnttnga nt
12910DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 9tcnnnntaca
101012DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 10ngtntcgtnc cg
121118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 11aacgggataa ctgcacct
181210DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 12tcaaggtaca
101310DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 13tgtaccttga
101412DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 14agacnttngn nt
121512DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 15gtntanttnn ag
121610DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 16ttgannttag
101710DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 17tgtannttga
101812DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 18ngtcgnnggu cu
121912DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 19ucggnntung nc
122010DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 20ctaaggtcaa
102110DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 21tcaaggtaca
102210DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 22ctaaggtcaa
102310DNAArtificial
SequenceDescription of Artificial Sequence Synthetic PNA
oligonucleotide which does not occur in nature 23tcnnggtaca
10